EP2526200A1 - Analyse in vivo pour quantifier l'activité de neurotoxine clostridienne - Google Patents

Analyse in vivo pour quantifier l'activité de neurotoxine clostridienne

Info

Publication number
EP2526200A1
EP2526200A1 EP11701015A EP11701015A EP2526200A1 EP 2526200 A1 EP2526200 A1 EP 2526200A1 EP 11701015 A EP11701015 A EP 11701015A EP 11701015 A EP11701015 A EP 11701015A EP 2526200 A1 EP2526200 A1 EP 2526200A1
Authority
EP
European Patent Office
Prior art keywords
clostridial neurotoxin
neurotoxin
test particle
reaction volume
clostridial
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP11701015A
Other languages
German (de)
English (en)
Other versions
EP2526200B1 (fr
Inventor
Thomas Wilk
Harold Taylor
Karl-Heinz Eisele
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merz Pharma GmbH and Co KGaA
Original Assignee
Merz Pharma GmbH and Co KGaA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merz Pharma GmbH and Co KGaA filed Critical Merz Pharma GmbH and Co KGaA
Priority to EP11701015.7A priority Critical patent/EP2526200B1/fr
Priority to SI201130831A priority patent/SI2526200T1/sl
Publication of EP2526200A1 publication Critical patent/EP2526200A1/fr
Application granted granted Critical
Publication of EP2526200B1 publication Critical patent/EP2526200B1/fr
Priority to HRP20160820TT priority patent/HRP20160820T1/hr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Definitions

  • This invention relates to novel methods for determining the unknown biological activity of a clostridial neurotoxin in a sample with respect to the known biological activity of a clostridial neurotoxin in a reference sample.
  • the method comprises the step of comparing the biological activity of a clostridial neurotoxin preparation with the biological activity of a standard preparation of a reference clostridial neurotoxin in certain in vitro systems.
  • botulinum neurotoxins have become the standard agent in the treatment of focal dystonias and spastic indications.
  • Treatment of patients generally involves injection of the neurotoxin into affected muscle tissue, bringing the agent near the neuromuscular end plate, i.e. close to the cellular receptor mediating its up- take into the nerve cell controlling said affected muscle.
  • Various degrees of neurotoxin spread have been observed. This spread is thought to correlate with the injected amounts and the particular preparation of neurotoxin injected. Resulting from the spread, systematic side effects caused by the inhibition of acetylcholine release, may be observed at nearby muscle tissue. The incidents of unintended paralysis of untreated muscles can largely be avoided by reducing the injected doses to the therapeutically relevant level.
  • Overdosing may also be a problem with regard to the patients' immune system, as the injected neurotoxin may trigger the formation of neutralizing antibodies. If this occurs, the neurotoxin will be inactivated without being able to relieve the involuntary muscle activity.
  • Differences in the dose equivalents or variations in the determined activity of preparations such as available sales products or batches produced during the manufacturing process, commonly a fermentation process, pose an increased risk for patients through possible side effects and the development of immunity. Therefore, it is of crucial importance to determine the biological activity of a clostridial neurotoxin contained in said sales products or production batches reliably (i.e. without significant variation) and as accurately as possible, in order to adjust the neurotoxin concentration to a reliable effective dose for the benefit of the patient. This may also serve as an incentive to the manufacturers to offer formulations allowing optimum exploitation of biological activity for different therapeutic purposes.
  • the botulinum neurotoxin testing is predominantly performed using the mouse LD 50 assay developed more than 40 year ago (see Boroff and Fleck, J. Bacteriol. 92 (1966) 1580-1581), which is accepted for potency testing by United States and European regulatory agencies.
  • This assay involves dosing mice with dilutions of the sample of botulinum neurotoxin being tested and calculating the dilution at which 50% of the mice would be expected to die. Since this bioassay requires up to 100 mice for testing a single sample, and takes up to four days to generate results, there is a large need for alternative methods that are faster and more accurate, and/or reduce, cause less pain, distress, and/or replace use of animals such as mice.
  • any such alternative method in order to be acceptable to regulatory agencies for the determination of potency, must be suitable for the intended purpose of the product in question and must be validated for sensitivity, specificity, reproducibility and robustness.
  • a suitable potency assay For botulinum neurotoxin testing, a suitable potency assay must be used to determine the dose of the final product or to compare the relative activities of different lots. Because botulinum neurotoxin activity is dependent upon three functional domains within the protein molecule, an acceptable potency assay must account for the activity of all domains.
  • a similar assay is based on the detection of SNAP25i 97 in differentiated Neuro-2A cells (Fernandez-Salas et al., ABS-29 presented at: Basic and Therapeutic Aspects of Botulinum and Tetanus Toxins International Conference (Toxins 2005), June 23- 25, 2005, Denver, CO; Steward et al., ABS-76 presented at: Basic and Therapeutic Aspects of Botulinum and Tetanus Toxins International Conference (Toxins 2005), June 23-25, 2005, Denver, CO).
  • cellular assays were set up that monitor the neuronal network or neurotransmitter release on clostridial neurotoxin treatment.
  • Chaddock et al. describe an assay based on the detection of potassium stimulated 3 H-glycine release in a primary culture of fetal rat spinal cord cells on treatment with BoNT/A (Chaddock et al., Protein Expr Purif. 2002; 25: 219-28).
  • Gross et al. used the reduction of spontaneous spiking and bursting as read-out in embryonic murine spinal cord and frontal cortex cells (Gross et al., Society for Neurosciences 2003, Abstract 122.9). That assay was originally developed by Keefer et al. using either BoNT/A or tetanus toxin (Keefer et al., Society for Neurosciences 2001 , Abstract 1302).
  • the present invention relates to an in vitro method for determining the biological activity of a preparation of a clostridial neurotoxin, comprising the steps of:
  • said at least one test particle comprises at least a membrane enclosing a reaction volume, wherein said membrane carries one or more receptors for said clos- tridial neurotoxin capable of mediating transfer of said clostridial neurotoxin to said reaction volume, and wherein said reaction volume comprises a substrate for the proteolytic activity of said clostridial neurotoxin.
  • the present invention also relates to a kit for an in vitro method for determining the biological activity of a preparation of a clostridial neurotoxin, comprising:
  • At least one test particle comprises at least a membrane enclosing a reaction volume, wherein said membrane carries one or more receptors for said clostridial neurotoxin capable of mediating transfer of said clostridial neurotoxin to said reaction volume, and wherein said reaction volume comprises a substrate for the proteolytic activity of said clostridial neurotoxin;
  • the term “in vitro” refers to a system that is not a living organism (i.e. not “in vivo”). Furthermore, an “in vitro” system does not include a functional multi-cellular part taken from such an organism, e.g. an organ or organ preparation.
  • the term “clostridial neurotoxin” refers to a natural neurotoxin obtainable from bacteria of the class Clostridia, including Clostridium tetani and Clostridium botulinum, or to a neurotoxin obtainable from alternative sources, including from recombinant expression or from genetic or chemical modification of such a natural neurotoxin.
  • the clostridial neurotoxins have endopeptidase activity.
  • Said neurotoxin may be obtained from a clostridial cell or by expression of the neurotoxin in a heterologous cell such as E. coli.
  • the term "preparation of a clostridial neurotoxin” refers to a composition comprising a clostridial neurotoxin, including, but not limited to, the raw material obtained from a fermentation process (supernatant, composition after cell lysis), a fraction comprising a clostridial neurotoxin obtained from separating the ingredients of such a raw material in a purification process, an isolated and essentially pure clostridial neurotoxin, and a formulation for pharmaceutical and/or aesthetic use comprising a clostridial neurotoxin and additionally pharmaceutically acceptable solvents and/or excipients.
  • the term "contacting" as used herein means exposing the test particle to the sample.
  • One way of exposing the test particle to the sample is to incubate (bathe) the test particle in the sample.
  • the test particle may be positioned e.g. in a reaction vessel containing a physiological buffer. The skilled person will realize that the choice of the buffer depends on the particularities of the test particle.
  • a suitable amount of the sample may be added to the reaction vessel.
  • the term "reference sample” refers to a sample taken from a standard preparation of a reference clostridial neurotoxin, i.e. from a preparation comprising a clostridial neurotoxin, where the biological activity of a given amount of such standard preparation is known.
  • the biological activity of such standard preparation may, for example, be determined by the mouse LD 50 assay described above.
  • the LD 50 value defines the quantity of neurotoxin at which 50% of a mouse population is killed if said quantity is applied to the mice of said mouse population.
  • the LD 50 value defines the so-called "unit” that is the commonly accepted unit used to define a quantity of a clostridial neurotoxin contained in a sample.
  • the method for determining said value is known to the person skilled in the art. The method is documented in the European Pharmacopoeia (6 th edition; available from European Directorate for the Quality of Medicines & HealthCare (EDQM), 7 allee Kastner, CS 30026, F-67081 France).
  • test particle refers to a particle that is characterized by at least an outer surface enclosing at least one cavity containing a reaction volume.
  • the outer surface of said test particle is a lipid bilayer enclosing a luminal reaction volume.
  • the particle may be generated de novo from its components or may be obtained from a cell by preparation methods known in the art (Gray, E. G. & Whittaker, V. P. (1962) J. Anat. (London) 96, 79-87).
  • test particles suitable for the method of the present invention may be obtained in the following manner: eighteen rats are decapitated, and the brains quickly removed.
  • the tissue (about 25 g, wet weight) is homogenized in 9 volumes of ice-cold 0.32 M sucrose in 3 mM Hepes- NaOH buffer, pH 7.4. The homogenate is centrifuged at 1 ,000 x g for 10 min. The supernatant is centrifuged at 10,000 x g for 40 min. The pellet is suspended in 9 volumes of 0.8 M sucrose in 3 mM Hepes-NaOH buffer, pH 7.4, and centrifuged at 80,000 x g for 90 min.
  • the pellet is washed twice by centrifugation at 10,000 x g for 10 min with 9 volumes of 3 mM Hepes-NaOH buffer, pH 7.0, containing 0.12 M NaC1 , 2.5 mM KCI, 2 mM MgCI 2 and 2 mM CaCI 2 , (HBS).
  • the washed pellet designated as the crude synaptosomal fraction, is disrupted in 2 volumes of 10 mM Hepes-NaOH buffer, pH 7.0, containing 1 mM EDTA (Dojindo Laboratories) and 1 mM dithiothreitol (Sigma) and then mixed with an equal volume of the same buffer containing 40 mM MEGA-9 (Dojindo).
  • protease inhibitors are phenylmethylsulfonyl fluoride (0.1 mM, Sigma), pepstatin A (5 pg/ml, Peptide Institute, Inc.), and calpain inhibitors I and II (3 pg/ml, Nacalai Tesque).
  • the diameter of such test particles is typically between 10 nm and 1 mm, particularly between 0.1 and 10 ⁇ .
  • the "test particle” is represented by a second test chamber separated from a first test chamber by a membrane, similar to the setup described in Simon & Blobel (A protein-conducting channel in the endoplasmic reticulum. Cell. 1991 May 3;65(3):371-80).
  • the first test chamber corresponds to the outer reaction volume, wherein said sample of said preparation of a clostridial neurotoxin is placed.
  • the membrane e. g. a lipid bilayer, corresponds to the surface of said test particle, and the second test chamber corresponds to the test particles inner cavity containing the reaction volume.
  • the sample is put in the first test chamber, said membrane carries one or more receptors for said clostridial neurotoxin capable of mediating transfer of said clostridial neurotoxin to said second chamber, and said second chamber comprises a substrate for the proteolytic activity of said clostridial neurotoxin.
  • reaction volume refers to the volume covered, and separated from the external volume, by the membrane surrounding the test particle.
  • reaction volume the reaction between the clostridial neurotoxin comprised in the sample of the clostridial neurotoxin preparation and the reaction partners for the clostridial neurotoxin, e.g. a substrate, or other reactions resulting in an observable and/or detectable biological effect may take place.
  • the reaction vol- ume is at physiological pH and osmolarity under reducing conditions, e.g. by the presence of glutathione, mercaptoethanol or DTT, and optionally ATP.
  • biological effect refers to change or modification of the status of the test particle that can be determined either directly or indirectly be observing one or more parameters associated with such change or modification.
  • changes or modifications are e.g. binding events, resulting in the formation of dimeric or multimeric structures, enzymatic reactions, such as proteolytic reactions, resulting in a decrease of substrates, and an increase in products resulting from the enzymatic reaction, such as one or more cleavage products from proteolysis, or changes to the release of signaling molecules, e.g. a neurotransmitter such as acetylcholine etc., from a test particle.
  • signaling molecules e.g. a neurotransmitter such as acetylcholine etc.
  • the biological effect of the sample containing the clostridial neurotoxin is compared to the effect of a reference sample.
  • the term "reference sample” refers to a preparation with a known quantity of neurotoxin.
  • the effect of the sample is compared to a standard dose-response curve established by measuring the effect of several reference samples, each containing a different amount of the reference neurotoxin.
  • the quantity of the reference sample i.e. the amount per standard unit, e.g. mg/ml, or mg/kg of body weight
  • the response is plotted on the Y axis.
  • the dose-response curve is typically sigmoidal, with the steepest portion in the middle, indicating the half-maximal response.
  • the response is typically expressed as percentage of the maximal response observed, so that the quantity causing the half-maximal response is the quantity causing a 50% response rate, e.g. 50% inhibition (IC 50 ), or 50% efficacy (EC 50 ).
  • the biological effect of the sample and the biological effect of the reference sample are determined in the same test particle.
  • the biological activity is determined by determining one or more of the following parameters: time-dependent decrease of the amount of unchanged starting substrate, time-dependent increase of the amount(s) of one or more cleavage products resulting from proteolytic cleavage of the substrate, time- dependent decrease of the amount of a compound, e.g. a neurotransmitter, released from the test particle.
  • the biological effect may be determined by using methods known in the art.
  • the amount of said substrate and said cleavage product may be determined in an immunoassay, e.g. in an ELISA assay capable discriminating between substrate and product.
  • release of said compound, e.g. neurotransmitter may be determined by HPLC methods or by labeling said compound, e.g. neurotransmitter, with an appropriate isotope or fluorophore followed by measuring radioactivity or fluorescence in the test particle and / or the surrounding medium (Jones RG, Liu Y, Sesardic D. (2009) J Immunol Methods. 343 21-7).
  • the term “membrane” refers to a layer that separates the outside of a test particle from the inside, where such layer is permissible, under appropriate conditions, for the clostridial neurotoxin, or components thereof.
  • a membrane may be in the form of a lipid bilayer, e.g. as in naturally occurring cellular structures, or as in artificially generated structures, such as liposomal structures. Accordingly and as explained hereinabove, the lipid bilayer may be assembled de novo from its components, e.g. phospolipids, gangliosides and receptors, or may be obtained from a cell, e.g.
  • cell derived membranous particles be used in the methods of the present invention and be supplemented e.g. by particular phospholipids or polypeptides. Moreover, in certain cases it may be necessary to treat said particles prior to their use with proteases in order to remove unwanted proteins.
  • the term "one or more receptors for said clostridial neurotoxin capable of mediating transfer of said clostridial neurotoxin to said reaction volume” refers to the receptors mediating cellular uptake of clostridial neurotoxin found in neuronal tissues susceptible for clostridial neurotoxin inhibition.
  • Known receptors used by neurotoxins in the natural context are either glycolipids (e.g. Couesnon et al. Cell Microbiol. 2008 Feb;10(2):375-87.
  • transmembrane proteins for example, human SV2 for botulinum neurotoxin serotype A, or synaptotagmins I and II for botulinum neurotoxin serotype B), or both (e.g. Rummel et al., Proc Natl Acad Sci U S A. 2007 Jan 2;104(1):359-64 and Dong et al., Science. 2006 Apr 28;312(5773):592-6).
  • the term "receptor” refers to any membrane resident capable of mediating uptake into the lumen of the test particle.
  • a phospholipid or a sugar or a polypeptide can be used as a receptor, as long as it is associated with the outer leaflet of the lipid bilayer and capable of mediating uptake of the neurotoxin into the lumen of the particle.
  • the present invention's teaching extends to methods employing e.g. GPI-anchored receptor fragments, i.e. GPI-anchored molecules comprising the neurotoxin binding site of a naturally occurring receptor such as SV2.
  • GPI-anchored receptor fragments i.e. GPI-anchored molecules comprising the neurotoxin binding site of a naturally occurring receptor such as SV2.
  • the test particle of the present invention is assembled de novo, it may be advantageous to replace receptor molecules having multiple membrane spanning domains with GPI-anchored analogs thereof.
  • the term “mediating transfer to said reaction volume” refers to the crossing of the light chain of the clostridial neurotoxin into the lumen of said test particle.
  • the method of the present invention is very sensitive if the receptor density on de novo assembled test particles is particularly high. Surprisingly, this effect is also observed with cell derived test particles supplemented with additional receptor molecules. Accordingly, it is envisaged by the inventors to optimize the test particles by adding receptor molecules up to the point of optimal sensitivity.
  • the term "substrate for the proteolytic activity of said clostridial neurotoxin” refers to any intracellular target for such clostridial neurotoxin (for example, human SNAP25 for botulinum neurotoxin serotype A, C and E, or Synaptobrevin for botulinum neurotoxin serotype B and F, and tetanus toxin).
  • targets are proteins, which are involved in the formation of a functional SNARE complex.
  • such substrate is inactivated by interaction with the neurotoxin, in another embodiment, such substrate is inactivated by proteolytic cleavage.
  • the substrate of the present invention's method may be a naturally occurring substrate such as the full length SNAP-25 polypeptide or a derivative thereof.
  • derivative refers for example to modified polypeptide substrates with N- terminal, C-terminal and/or internal truncations or additions, and to modified polypeptides comprising one or more amino acid additions, substitutions and/or deletions.
  • Said polypeptide substrate may be of human origin or may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the human polypeptide sequence.
  • said substrate is labeled with one or more labels.
  • the label may be for example a fluorescent label or a mixture of different fluorescent labels or any other detectable tag, including e.g. radioactive labels, DNA / RNA labels, Peptide or protein labels (GST, MBP, NusA, Ubiquitin etc.), enzyme labels (AP, HRP, Luciferase etc..) or physical labels such as non-radioactive isotopes.
  • said method further comprises after step (a) the step of:
  • the term "decreasing the pH value” as used herein means lowering the pH from pH 7.4 to a pH value below 6.5, particularly below pH 6, more particularly below pH 5 and more particularly between 4 and 5, and most particularly to about 4.5.
  • Conditions that emulate the pH and redox gradient across endosomes, for example pH 5.3 on the cis-compartment containing BoNT and pH 7.0 on the trans-compartment supplemented for example with the membrane nonpermeable reductant Tris-(2- carboxyethyl) phosphine (TCEP) support channel activity.
  • said method further comprises after step (a) the step of:
  • such adjustment(s) is/are performed in order to allow to allow said proteolytic activity, i.e. the cleavage of the substrate by the light chain of the clostridial neurotoxin in the lumen, i.e. the reaction volume of the test particle.
  • step (b) comprises the step of:
  • binding is determined by using binding molecules, such as antibodies, directed against the clostridial neurotoxin (i.e. direct determination of the amount of bound clostridial neurotoxin), or by using binding molecules, such as antibodies, directed against a receptor for the clostridial neurotoxin (i.e. indirect determination of the amount of bound clostridial neurotoxin by determining the amount of free, i.e. unbound receptors).
  • binding molecules such as antibodies directed against the clostridial neurotoxin
  • binding molecules such as antibodies, directed against a receptor for the clostridial neurotoxin
  • neurotoxin is determined e.g. in an immunoassay (e.g. ELISA, Western Blot).
  • an immunoassay e.g. ELISA, Western Blot
  • step (b) comprises the step of:
  • proteolysis is determined by ELISA or SNAPtide® methods.
  • ELISA methods can be employed that use antibodies directed against either the uncleaved substrate or one or more of the cleavage products (see, for example, Ekong et al., Microbiology 1997; 143: 3337-47).
  • the term "SNAPtide® methods" as used herein refers to an assay format which is based on the fluorometric measurement of botulinum toxin activity by providing fluorescently-labeled SNAP25- based substrates containing the native cleavage site for botulinum neurotoxin of serotype A (SNAPtide®), or of serotype E (SNAP Etide®).
  • SNAPtide® and SNAP Etide® assays are available e.g. from List Biological Laboratories, Inc..
  • the test particle is a test particle obtainable from a cell, in particular a non-cellular test particle, such as subcellular membrane fragments or erythrocyte ghosts which naturally or artificially contain appropriate receptors for the analyte.
  • the non-cellular test particle is a synapto- some.
  • the synaptosome is derived from a mammalian brain e.g. a rat or mouse brain, or a brain from a transgenic animal expressing one or more human SNARE proteins.
  • said non-cellular test particle is an artificially generated particle.
  • the artificially generated particle may for example be a liposome.
  • Such particle may be assembled essentially as described in: Liposome Drug Delivery Systems, G. V. Betageri, S. A. Jenkins, D. L. Parsons (1993).
  • the test particle comprises a) a membrane e.g., a spherical phospholipide bilayer, enclosing an inner reaction volume.
  • an adequate buffer system e.g. 50 mM HEPES / 20 ⁇ ZnCI 2 pH 7.0
  • a reducing agent e.g. 5 mM Dithiothreitol
  • clostridial neurotoxin c a proteolysis substrate for the clostridial neurotoxin c
  • the receptor binding sites e.g., the receptors of clostridial neurotoxin Type A, i.e. the synaptic vesicle protein 2 (SV2) and the ganglioside GT1 b.
  • these receptors are integrated into the membrane of the test particle exposing their targeting structures to the outer surface of the test particle.
  • the clostridial neurotoxin referred to herein is a neurotoxin of C. botulinum and is of serotype A, B, C1 , D, E, F or G or is a biologically active derivative thereof, wherein said derivative may be a genetically modified neurotoxin such as a mutant comprising a deletion of one or more amino acids, an addition of one or more amino acids and/or a substitution of one or more amino acids.
  • said deleted or added amino acids are consecutive amino acids. According to the teaching of the present invention, any number of amino acids may be added or deleted, as long as the neurotoxin is biologically active.
  • a derivative of the neurotoxin may be a biologically active fragment of a naturally occurring neurotoxin. This neurotoxin fragment may contain an N-terminal, C-terminal and/or one or more internal deletion(s).
  • Bioly active means, said derivative is taken up into the nerve cell and is capable of denervating said nerve from the muscle or gland, to which it is connected.
  • said clostridial neurotoxin may be associated with complexing proteins or may be free of complexing proteins (see below).
  • the clostridial neurotoxin is of serotype A or E, and/or is a biologically active derivative thereof.
  • botulinum neurotoxin refers to neurotoxins obtainable from Clostridium botulinum.
  • serotypes A, B, C1 , D, E, F and G are known, including certain subtypes (e.g. A1 , A2, and A3).
  • these neurotoxins are formed as protein complexes comprising a neurotoxic component and at least another non-toxic protein.
  • botulinum neurotoxin serotype A refers to the botulinum neurotoxin complex, such as the 450 kDa and the 900 kDa complexes which are e.g. obtainable from cultures of Clostridium botulinum. Preparations comprising such complexes are commercially available, e.g. from Ipsen Ltd. (Dysport®) or Allergan Inc. (Botox®).
  • botulinum neurotoxin also refers to botulinum neurotoxin which is free of complexing proteins and which has a molecular weight of approximately 150kDa.
  • the clostridial neurotoxin is an iso- lated neurotoxic component of botulinum neurotoxin, particularly of botulinum neurotoxin of serotype A or E.
  • isolated neurotoxic component of botulinum neurotoxin serotype refers to the neurotoxic component of the corresponding botulinum neurotoxin serotype complex free of complexing proteins.
  • This neurotoxic component is a two-chain polypeptide with a 100 kDa heavy chain joined by a disulfide bond to a 50 kDa light chain.
  • the heavy chain is responsible for entry into the neuronal cell, while the light chain comprises an endopeptidase activity responsible for cleaving one or more proteins that is/are part of the so-called SNARE-complex involved in the process resulting in the release of neurotransmitter into the synaptic cleft.
  • a high purity neurotoxic component of the neurotoxin of C. botulinum of serotype A, free of any other botulinum protein, is e.g. available from Merz Pharmaceuticals GmbH, Frankfurt (Xeomin ® ).
  • other isolated fragment of any said botulinum neurotoxin where such fragment has neurotoxic activity refers to a neurotoxin complex that lacks certain of the non-toxic proteins of a botulinum neurotoxin as defined above, or certain parts of the neurotoxic component as defined above comprised therein, while maintaining the neurotoxic activity.
  • other isolated fragment of any said ... neurotoxic component, where such fragment has neurotoxic activity refers to a neurotoxin component that lacks certain certain parts of the neurotoxic component of a botulinum neurotoxin as defined above, while maintaining the neurotoxic activity.
  • the term “derivative” and the term “variant or synthethic analogue” refer to a neurotoxin complex or neurotoxic component that is a chemically, enzymatically or genetically modified derivative of a neurotoxin as defined herein, including chemically or genetically modified neurotoxin from C. botulinum.
  • a chemically modified derivative may be one that is modified by pyru- vation, phosphorylation, sulfatation, lipidation, pegylation, glycosylation and/or the chemical addition of an amino acid or a polypeptide comprising between 2 and about 100 amino acids .
  • An enzymatically modified derivative is one that is modified by the activity of enzymes, such as endo- or exoproteolytic enzymes.
  • a genetically modified derivative is one that has been modified by deletion or substitution of one or more amino acids contained in, or by addition of one or more amino acids (including polypeptides comprising between 2 and about 100 amino acids) to, the proteins of said neurotoxin or a neurotoxic component thereof.
  • Methods for making such chemically or genetically modified derivatives, and methods for identifying whether such derivatives maintain neurotoxic activity are well known to anyone of ordinary skill in the art.
  • said clostridial neurotoxin is selected from the group of botulinum neurotoxin serotype A or E, the isolated neurotoxic component of botulinum neurotoxin serotype A or E, an isolated fragment of botulinum neurotoxin serotype A or E, or of the neurotoxic component of botulinum neurotoxin serotype A or E, where such fragment has neurotoxic activity, and any variant or synthetic analogue of botulinum neurotoxin serotype A, or E, respectively, or of the neurotoxic component thereof, where such variant or analogue has neurotoxic activity.
  • fragment relates to a neurotoxin lacking 1 , 2, 3, 4, 5, up to 10, up to 15, up to 25, up to 50, up to 100, up to 200, up to 400, up to 500 amino acids or even more amino acids.
  • said amino acids are consecutive amino acids.
  • said clostridial neurotoxin is the neurotoxin of C. botulinum of serotype A, or is the isolated neurotoxic component of botulinum neurotoxin of serotype A, which is available from Merz Pharmaceuticals GmbH, Frankfurt (Xeomin ® ).
  • said clostridial neurotoxin preparation is for pharmaceutical and/or aesthetic use.
  • the clostridial neurotoxin in the clostridial neurotoxin preparation is produced in a fermentation process.
  • the clostridial neurotoxin preparation is produced by a fermentation process using Clostridium botulinum strains.
  • Methods for the fermentation process using Clostridium botulinum strains are well known in the art (see, for example, Siegel and Metzger, Appl. Environ. Microbiol. 38 (1979) 606-611 ; Siegel and Metzger, Appl. Environ. Microbiol. 40 (1980) 1023-1026).
  • the clostridial neurotoxin preparation is produced in heterologous cells, i.e. it is produced recombinantly by expressing nucleic acid sequences encoding a neurotoxin in an appropriate host cells.
  • Methods for the recombinant expression of clostridial neurotoxin are well known in the art (see, for example, WO 2006/076902 or EP 1 206 554).
  • An example of a heterologous cell is E. coli.
  • the clostridial neurotoxin in said clostridial neurotoxin preparation and the reference clostridial neurotoxin belong to the same serotype.
  • the clostridial neurotoxin in said clostridial neurotoxin preparation and the reference clostridial neurotoxin are identical.
  • the terms “belong to the same serotype” and “identical” refer to the nature of the active neurotoxic agent present in the clostridial neurotoxin preparation and the reference clostridial neurotoxin.
  • the clostridial neurotoxin preparation contains the isolated neurotoxic component (i.e. without any com- plexing proteins) of botulinum neurotoxin serotype A
  • the reference clostridial neurotoxin is botulinum neurotoxin serotype A (i.e.
  • the clostridial neurotoxin in said clostridial neurotoxin preparation and the reference clostridial neu- rotoxin belong to the same serotype, but are not identical.
  • the clostridial neurotoxin preparation contains the isolated neurotoxic component (i.e.
  • the reference clostridial neurotoxin is the isolated neurotoxic component of botulinum neurotoxin serotype A as well, the clostridial neurotoxin in said clostridial neurotoxin preparation and the reference clostridial neurotoxin are identical (even though the clostridial neurotoxin preparation and the preparation containing the reference clostridial neurotoxin may differ in the amount and concentration of the respective neurotoxin and/or the presence of additional ingredients such as buffers, excipients etc.).
  • the clostridial neurotoxin is of serotype A
  • the one or more receptors are taken from the list of: SV2, a ganglioside receptor, and a biologically active derivative of SV2 or a ganglioside receptor.
  • the ganglioside receptor is taken from the list of GT1 b (see Rummel et al., Molecular Microbiology 2004; 51 : 631-43) and GD1b.
  • the substrate is SNAP-25 or a biologically active variant thereof.
  • a preparation of rat brain synaptosomes is obtained as described in the literature from a Percoll gradient after homogenization of rat brain (see Rummel et al., Molecular Microbiology 2004; 51 : 631-43).
  • the synaptosomes are diluted in physiological buffer (e.g. 140 mM NaCI, 5 mM KCI, 1 mM MgCI 2 , 1 mM CaCI 2 , 20 mM Hepes, 10 mM glucose, 0.5% BSA, pH 7.4).
  • physiological buffer e.g. 140 mM NaCI, 5 mM KCI, 1 mM MgCI 2 , 1 mM CaCI 2 , 20 mM Hepes, 10 mM glucose, 0.5% BSA, pH 7.4
  • the amount of synaptosomes being present is determined by determining the total amount of synaptosomal proteins. For all experiments, a fixed concentration of total synaptosomal proteins, e.g. 10 mg/ml, is
  • a fixed amount of different dilutions from a dilution series of a reference botulinum neurotoxin serotype A with known biological activity is added to the synaptosome preparation.
  • the synaptosomes are repeatedly washed using physiological buffer and centrifugation, in order to remove unbound neurotoxin.
  • the pH of the solution to pH 4.5 by adding, for example, 1 M HCI.
  • Incubation is continued for about 30 min.
  • Synaptosomes are lysed using, for example, Triton X-100 (see Sahyoun et al., J. Biol. Chem.
  • SNAP25 and/or its cleavage products are determined in an ELISA assay as described (Ekong et al., Microbiology 1997; 143: 3337-47).
  • a botulinum neurotoxin inhibitors such as described, for example, by Boldt et al., Chem. Commun., 2006, 3063-3065, can be added to terminate the proteolytic activity of the neurotoxin.
  • Clostridium neurotoxin preparation in otherwise identical fashion.
  • the amount of Clostridium neurotoxin preparation necessary to achieve EC 50 i.e. the half-maximal cleavage of the SNAP25 substrate
  • the amount of Clostridium neurotoxin preparation necessary to achieve EC 50 can then be correlated to the corresponding amount of reference botulinum neurotoxin serotype A with known biological activity.
  • Liposomes are formed from phospholipids, such as phosphatidylethanola- mine, phosphatidylcholine, or mixtures thereof, as described in the literature, in the presence of recombinant SV2, GT1b and/or GD1b, and SNAPtide® (o-Abz/Dnp) or SNAPtide® (FITC/DABCYL) (List Biological Laboratories, Inc., Product Nos. 520 and 521 , respectively). o-Abz/Dnp). The resulting liposomes are repeatedly washed in order to remove the excess of reactants not incorporated into the liposomes. The amount of SV2 integrated into the membranes can be determined by using antibodies directed against SV2 (Santa Cruz Biotechnology, Inc.).
  • a fixed amount of different dilutions from a dilution series of a reference botulinum neurotoxin serotype A with known biological activity (expressed as mouse units) is added to the liposome preparation. After incubation for about 2 h at about 0°C, the liposomes are repeatedly washed using physiological buffer and centrifuga- tion, in order to remove unbound neurotoxin. Thereafter, the pH of the solution to pH 4.5 by adding, for example, 1 M HCI. Incubation is continued for about 30 min, and the increase in fluorescence intensity is being determined (see instruction manual for the SNAPtide® assay), which is directly proportional to the amount of cleavage that is occurring.
  • the cleavage reaction can directly be observed inside the test particle by using fluorescence correlation spectroscopy (see, for example, Brock and Jovin (1998). Fluorescence correlation microscopy (FCM) - fluorescence correlation spectroscopy (FCS) taken into the cell. Cell. Mol. Biol. 44, 847-856).
  • fluorescence correlation microscopy FCM
  • FCS fluorescence correlation spectroscopy
  • Clostridium neurotoxin preparation in otherwise identical fashion.
  • the amount of Clostridium neurotoxin preparation necessary to achieve EC 5 o i e. the half-maximal cleavage of the SNAPtide® substrate
  • the amount of Clostridium neurotoxin preparation necessary to achieve EC 5 o can then be correlated to the corresponding amount of reference botulinum neurotoxin serotype A with known biological activity.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne de nouveaux procédés de détermination de l'activité biologique inconnue d'une neurotoxine clostridiale dans un échantillon par rapport à l'activité biologique connue d'une neurotoxine clostridiale dans un échantillon de référence. Ces nouveaux procédés comprennent l'étape de comparaison de l'activité biologique d'une préparation de neurotoxine clostridiale à l'activité biologique d'une préparation standard d'une neurotoxine clostridiale de référence dans certains systèmes in vitro.
EP11701015.7A 2010-01-22 2011-01-19 Analyse in vivo pour quantifier l'activité de neurotoxine clostridienne Active EP2526200B1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP11701015.7A EP2526200B1 (fr) 2010-01-22 2011-01-19 Analyse in vivo pour quantifier l'activité de neurotoxine clostridienne
SI201130831A SI2526200T1 (sl) 2010-01-22 2011-01-19 In vitro preizkus za kvanitificiranje aktivnosti klostridijskega nevrotoksina
HRP20160820TT HRP20160820T1 (hr) 2010-01-22 2016-07-06 In vitro test za kvantificiranje aktivnosti klostridijskog neurotoksina

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US33650210P 2010-01-22 2010-01-22
EP10000667 2010-01-22
EP11701015.7A EP2526200B1 (fr) 2010-01-22 2011-01-19 Analyse in vivo pour quantifier l'activité de neurotoxine clostridienne
PCT/EP2011/000204 WO2011088993A1 (fr) 2010-01-22 2011-01-19 Essai in vitro de quantification de l'activité de la neurotoxine clostridiale

Publications (2)

Publication Number Publication Date
EP2526200A1 true EP2526200A1 (fr) 2012-11-28
EP2526200B1 EP2526200B1 (fr) 2016-04-06

Family

ID=42079153

Family Applications (1)

Application Number Title Priority Date Filing Date
EP11701015.7A Active EP2526200B1 (fr) 2010-01-22 2011-01-19 Analyse in vivo pour quantifier l'activité de neurotoxine clostridienne

Country Status (8)

Country Link
US (1) US9310386B2 (fr)
EP (1) EP2526200B1 (fr)
ES (1) ES2576059T3 (fr)
HR (1) HRP20160820T1 (fr)
HU (1) HUE029697T2 (fr)
PL (1) PL2526200T3 (fr)
SI (1) SI2526200T1 (fr)
WO (1) WO2011088993A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2266600B1 (fr) * 2004-07-26 2014-09-10 Merz Pharma GmbH & Co. KGaA Composition thérapeutique à base de neurotoxine botulique
AR061669A1 (es) * 2006-06-29 2008-09-10 Merz Pharma Gmbh & Co Kgaa Aplicacion de alta frecuencia de terapia con toxina botulinica
US9161970B2 (en) * 2007-12-12 2015-10-20 Allergan, Inc. Dermal filler
ES2576059T3 (es) * 2010-01-22 2016-07-05 Merz Pharma Gmbh & Co. Kgaa Ensayo in vitro para cuantificar la actividad de neurotoxina clostridial
CA2801421A1 (fr) * 2010-06-11 2012-04-12 Synaptic Research, Llc Procedes d'indication d'activite de protease de regle n-terminale
EP2734635A1 (fr) * 2011-07-19 2014-05-28 ETH Zürich Moyens et procédés pour déterminer la présence de neurotoxines clostridiales
US20140287433A1 (en) * 2011-07-19 2014-09-25 ETH Zürich Means and methods for determining clostridial neurotoxins

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2277854T5 (es) 1999-08-25 2011-02-04 Allergan, Inc. Neurotoxinas recombinantes activables.
US6504006B1 (en) * 2001-10-12 2003-01-07 Nancy Rose Shine Substrate peptides and assays for detecting and measuring proteolytic activity of serotype A neurotoxin from clostridium botulinum
DE102005002978B4 (de) 2005-01-21 2013-04-25 Merz Pharma Gmbh & Co. Kgaa Rekombinante Expression von Proteinen in einer disulfidverbrückten, zweikettigen Form
ES2576059T3 (es) * 2010-01-22 2016-07-05 Merz Pharma Gmbh & Co. Kgaa Ensayo in vitro para cuantificar la actividad de neurotoxina clostridial

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2011088993A1 *

Also Published As

Publication number Publication date
US9310386B2 (en) 2016-04-12
US20130022992A1 (en) 2013-01-24
ES2576059T3 (es) 2016-07-05
PL2526200T3 (pl) 2016-10-31
SI2526200T1 (sl) 2016-07-29
EP2526200B1 (fr) 2016-04-06
HRP20160820T1 (hr) 2016-08-12
WO2011088993A1 (fr) 2011-07-28
HUE029697T2 (en) 2017-03-28

Similar Documents

Publication Publication Date Title
EP2526200B1 (fr) Analyse in vivo pour quantifier l'activité de neurotoxine clostridienne
US11879141B2 (en) Nucleic acid molecules encoding clostridium histolyticum collagenase II and methods of producing the same
Pellett Progress in cell based assays for botulinum neurotoxin detection
Shibatani et al. Proteomic analysis of mammalian oligosaccharyltransferase reveals multiple subcomplexes that contain Sec61, TRAP, and two potential new subunits
Whitemarsh et al. Characterization of botulinum neurotoxin A subtypes 1 through 5 by investigation of activities in mice, in neuronal cell cultures, and in vitro
Adler et al. Persistence of botulinum neurotoxin A demonstrated by sequential administration of serotypes A and E in rat EDL muscle
US8492109B2 (en) Methods for the delivery of toxins or enzymatically active portions thereof
Slakeski et al. C-terminal domain residues important for secretion and attachment of RgpB in Porphyromonas gingivalis
TW201639877A (zh) 工程化之肉毒桿菌神經毒素
TW201839132A (zh) 製造經蛋白分解處理之多肽之方法
JP6670842B2 (ja) 細胞内へのボツリヌス神経毒素の特異的取り込みを強化するための方法
Pellett et al. Sensitive and quantitative detection of botulinum neurotoxin in neurons derived from mouse embryonic stem cells
Kawaguchi et al. Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor
Nakamura et al. Unique biological activity of botulinum D/C mosaic neurotoxin in murine species
WO2013011055A1 (fr) Moyens et procédés pour déterminer la présence de neurotoxines clostridiales
JP2007516257A (ja) 脂質ラフツおよびクロストリジウム毒素
Sapra et al. Proteomic analyses of murine macrophages treated with Bacillus anthracis lethal toxin
Ji et al. SNAP-25, a SNARE protein, inhibits two types of K+ channels in esophageal smooth muscle
US20140287433A1 (en) Means and methods for determining clostridial neurotoxins
Selvan et al. Localization and characterization of prolipoprotein diacylglyceryl transferase (Lgt) critical in bacterial lipoprotein biosynthesis
Lei et al. Soluble oligomers of the intramembrane serine protease YqgP are catalytically active in the absence of detergents
Yadava et al. Import and orientation of the MWFE protein in mitochondrial NADH-ubiquinone oxidoreductase
US20240218326A1 (en) Novel neuronal cell line for measuring titer of neurotoxin
Lilla et al. Validation of recombinant and bovine chymosin by mass spectrometry
Foran Characterisation of the Endoproteinase Activities of Tetanus and Botulinum B Neurotoxins and Their Use in the Study of Exocytosis

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20120813

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20151014

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 787891

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160415

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: JACOBACCI AND PARTNERS SA, CH

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602011024858

Country of ref document: DE

REG Reference to a national code

Ref country code: EE

Ref legal event code: FG4A

Ref document number: E011834

Country of ref document: EE

Effective date: 20160427

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2576059

Country of ref document: ES

Kind code of ref document: T3

Effective date: 20160705

REG Reference to a national code

Ref country code: HR

Ref legal event code: TUEP

Ref document number: P20160820

Country of ref document: HR

REG Reference to a national code

Ref country code: NL

Ref legal event code: FP

REG Reference to a national code

Ref country code: HR

Ref legal event code: T1PR

Ref document number: P20160820

Country of ref document: HR

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160806

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160706

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160808

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160707

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602011024858

Country of ref document: DE

REG Reference to a national code

Ref country code: HR

Ref legal event code: ODRP

Ref document number: P20160820

Country of ref document: HR

Payment date: 20170112

Year of fee payment: 7

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 7

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

26N No opposition filed

Effective date: 20170110

REG Reference to a national code

Ref country code: HU

Ref legal event code: AG4A

Ref document number: E029697

Country of ref document: HU

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: EE

Payment date: 20170119

Year of fee payment: 7

Ref country code: LT

Payment date: 20170105

Year of fee payment: 7

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: LV

Payment date: 20170123

Year of fee payment: 7

Ref country code: HR

Payment date: 20170112

Year of fee payment: 7

Ref country code: BE

Payment date: 20170124

Year of fee payment: 7

Ref country code: PL

Payment date: 20170116

Year of fee payment: 7

Ref country code: SI

Payment date: 20170105

Year of fee payment: 7

Ref country code: HU

Payment date: 20170104

Year of fee payment: 7

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170119

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 8

REG Reference to a national code

Ref country code: HR

Ref legal event code: PBON

Ref document number: P20160820

Country of ref document: HR

Effective date: 20180119

REG Reference to a national code

Ref country code: LT

Ref legal event code: MM4D

Effective date: 20180119

REG Reference to a national code

Ref country code: EE

Ref legal event code: MM4A

Ref document number: E011834

Country of ref document: EE

Effective date: 20180131

REG Reference to a national code

Ref country code: AT

Ref legal event code: UEP

Ref document number: 787891

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160406

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170119

REG Reference to a national code

Ref country code: SI

Ref legal event code: KO00

Effective date: 20180925

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180120

Ref country code: LT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180119

Ref country code: EE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180131

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

REG Reference to a national code

Ref country code: BE

Ref legal event code: FP

Effective date: 20160704

Ref country code: BE

Ref legal event code: MM

Effective date: 20180131

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180119

Ref country code: LV

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180119

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180131

Ref country code: SI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180120

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160406

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

Ref country code: PL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180119

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230506

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20240123

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: ES

Payment date: 20240216

Year of fee payment: 14

Ref country code: IE

Payment date: 20240118

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: AT

Payment date: 20240118

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20240119

Year of fee payment: 14

Ref country code: GB

Payment date: 20240124

Year of fee payment: 14

Ref country code: CH

Payment date: 20240202

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20240131

Year of fee payment: 14

Ref country code: FR

Payment date: 20240124

Year of fee payment: 14